1. Field of the Invention
The present invention is related to plastic vessels for thermal cycling applications. In particular, the present invention provides plastic vessels with ultra thin walls allowing for efficient heat transfer in apparatus for thermal cycling applications as well as methods and instruments for the manufacture thereof.
2. Description of Related Art
A typical thermal cycling procedure is the performance of the polymerase chain reaction (PCR). Generally, the purpose of PCR is production (or amplification) of larger volumes of DNA which are identical in chemical makeup to minute volumes of native DNA obtained from limited sources. PCR is performed by utilizing a thermally controlled enzymatic reaction to make identical copies of double stranded DNA found in minute initial samples and then using those copies to generate other identical copies in successive cycles. Ideally each cycle will double the amount of DNA present. An extension of PCR for amplification is an analytical method known to those skilled in the art as quantitative PCR (also known as real time PCR) This method involves the real time monitoring of the amount of DNA product produced in each successive amplification cycle.
Generally speaking, PCR requires that the sample to be amplified, along with the other reaction components, be sealed into a reaction vessel and be incubated at two different temperatures, the first being for primer hybridization and primer extension reaction and the second being for denaturation. The latter step comprises separation of the double stranded DNA into single strand templates for use in the next successive hybridization and extension cycle. Typical temperature cycles require that the reaction mix be held with great accuracy (+/−0.3 degrees C.) at each incubation temperature for prescribed periods of time and that the identical cycle or one substantially similar be repeated many times, typically as many as 30. The incubation temperatures of the steps within a given cycle may range from 94 degrees C. to 37 degrees C.
It is desirable to change from temperature to temperature within a given cycle as rapidly as possible primarily for two reasons:                1. The enzymatic reaction has optimal temperatures for each step and strict adherence to those temperatures results in far more efficient DNA amplification.        2. The length of a given cycle is determined by the need for the reaction mixture to be held at each incubation temperature for a specified period time. Time spent transitioning between temperatures adds to the cycle time and, given the large number of cycles required, typically about 30, contributes greatly to the overall time required to complete the PCR process.        
This having been said, the fact remains that as regards the elapsed time to perform a set of laboratory protocol steps, in which PCR is one of the those steps, PCR will be the rate limiting step. Thus, a primary objective of those familiar with the process is to decrease the overall time required to perform PCR.
Since the reaction mix is an aqueous solution, as the high reaction temperatures, near the boiling point of water, are encountered, a portion of the reaction mix goes into vapour phase and a pressure increase inside the sealed sample tube is induced. Since a loss of the vapour from the sealed sample vessel would result in a change of concentration of the reaction mix components, producing the potential effect of a PCR reaction failure and loss of precious sample, it is imperative that the method of sealing the reaction vessels be robust.
Typically, temperature cycling of the vessels containing the reaction mix for the PCR reaction has been accomplished in one of two ways:                1. The more widely utilized method is to use a thermal cycling instrument comprised in part of a temperature controlled, highly thermally conductive sample block having wells of a geometry matching that of the portion of the disposable reaction vessel which contains the reaction mix. The geometric match of the mating areas of the vessel and the block provide intimate contact and a resultant thermal system consisting essentially of three components: the instrument's sample block/associated heat pump components, the vessel which contains the reaction mix (typically moulded plastic) and the reaction mix itself.                    The minimum cycle time limitation of PCR performed using these instruments, has, in general, been the thermal mass of the sample block and related heat pump components of the instrument system. Recent technical improvements in some commercially available instruments have decreased the cycle time of the instrument's sample block such that the thermal mass of conventional moulded plastic PCR vessels may be defined as the rate limiting factor for thermal transfer to the reaction sample itself, e.g. the rate limiting factor of the minimum overall time required for PCR.                        2. The second, and less encountered, method is to use an instrument consisting of several fixed temperature liquid baths, typically water, between which the vessels are automatically moved according to the dictates of the PCR protocol. This method is typically encountered in a high throughput situation where it is desirable to process many vessels simultaneously.                    Of course, in this case the vessel comes in intimate contact with the liquid so, again, the thermal mass of conventional plastic thin walled PCR vessels may be defined as the rate limiting factor for thermal transfer to the reaction sample itself, e.g., the rate limiting factor of the minimum overall time required for the total PCR protocol.                        
There are a number of commercially available thermal cycling vessels and accompanying sealing systems. The vessels are available in a number of formats including single tubes, tubes arranged in tray type arrays, typically known as multi-well plates, compatible with automated laboratory equipment and also strips of attached tubes, typically arranged with a tube center to center distance matching that found in one dimension of the multi-well plates. The single tubes and strips of tubes are generally sealed by means of a moulded cap or strip of caps that fit securely into the mouth of the tube(s). These caps may be separate or integral to the tube(s) It is reasonable to say that the single tubes and strips of tubes are produced exclusively via typical high pressure injection moulding processes.
The multi-well plate format has become the preferred format for many thermal cycling applications. Currently there are two methods of producing the multi-well plates. One of the early, and still utilized, methods of manufacture is via conventional thermoforming methods in which a sheet of plastic compatible with this manufacturing technique is heated to the softening point and forced to conform to the shape of a form which is the negative of the geometry of the multiwell plate. This method has the advantage of producing a low cost part with low thermal mass and very thin tube wall thicknesses. Such a product is offered in a 96 tube format by the Corning-CoStar company of Kennebunk, Me.
Unfortunately the drawbacks of this method result in a part unsuitable for nearly all latter day applications for several reasons:                1. Since the raw material is a thin sheet of plastic, the resultant multi-well plate has little structural integrity and no compatibility with automated plate handling equipment due to lack of rigidity.        2. The thermo-forming technique is a very imprecise process which offers no control over cross-sectional thinning as the softened plastic is drawn into the shape of the part. As a result, the concentricity of the formed tubes diameter is extremely poor. An examination of typical commercially available multi-well plates revealed wall thickness dimensions within given wells with variations as much as 6× (0.001″ to 0.006″).        3. A narrow selection of materials is available for use in the thermo-forming process. Few of these are optimal for use in PCR protocols and those that are, are not optimal for the thermo-forming process. Typical commercially available thermoformed plates are manufactured of polycarbonate sheet stock.        4. The thermoforming process is not conducive to forming small, consistently well defined features such as raised geometry around mouths of tubes, for instance to improve sealing performance.        
Due to the limitations cited with respect to thermoformed plates, high-pressure injection moulding has become the preferred method of manufacture for multi-well plates. High-pressure injection moulding has allowed the introduction of multi-well plates with many desirable features such as:                1. Tube wall thickness on the order of 0.009″ (2.3 mm) which is suitable for use in many thermal cycling protocols performed in standard thermal cycling equipment.        2. Increased rigidity such as is found in the HardShell multi-well plate from MJ Research/Biorad or the Twin Tec multi-well plate from Eppendorf, both produced by injection moulding processes.        3. Ability to form precise geometric features such as raised rims around the mouths of tubes which permit secure sealing using a wide variety of sealing systems such as films which may be heat sealed to the rims, elastomeric pads which form a very effective gasket seal against the raised rims of the multi-well plate when sealing pressure is applied over the pad surface.        4. A variety of materials are compatible with high-pressure injection moulding.        
Commercially available injection moulded sample vessels in various formats for use in thermal cycling are currently offered by a number of companies. Several injection moulded thermal cycling vessels are currently marketed as “thin wall” and include products available from Applera Corporation and from MJ Research/BioRad Laboratories of Hercules, Calif. The BioRad Laboratories product is marketed under MJ Research's trade name “HardShell” These are typically touted as having “thin” walls with typical nominal thickness of the thin wall section of between 0.009 inch and 0.015 inch (0.23 mm to 0.38 mm). One such product, in the format of a single tube with attached cap, is described in Published US Patent Application No. 2005/0084957, dated 21 Apr. 2005 and assigned to Applera Corporation of Foster City, Calif. Similar products have been commercially available for a number of years.
In any event, thermal cycling vessels with wall sections in this dimensional range do not approach the reduction in wall thickness required to take full advantage of the faster thermal cycling capabilities of the latest instrument technology. Further, careful examination of the thin wall areas of these commercially available injection moulded PCR vessel tubes will often reveal a lack of concentricity between the inner and outer diameters of the thin wall sections of the tubes. In the case of some micro-titer plates differences of 2× or greater may be observed. This is undesirable because it leads to uneven heat transfer to the reagent sample and consequently a less efficient PCR reaction.
As the previous narrative illustrates, the technology of thermal cycling instrumentation is advancing to the point that the thermal cycling instrumentation is no longer the rate limiting factor in thermal cycling. Rather, conventional thermal cycling vessels become the rate limiting step in the performance of PCR. Therefore there is an emerging need for a new multi-well plate design and method of manufacture, which affords the benefits of manufacture by the injection moulding process but possesses thinner tube walls than manufacture by currently known injection moulding methods will allow.
One German company, Analytic Jena, currently offers an alternative style ultra thin wall plate for PCR. It is a sort of hybrid which is formed by joining two components using two different manufacturing processes, e.g. tubes are formed by thermoforming/vacuum forming the tube shapes from polypropylene sheet into an array of 36 holes preformed in an injection moulded superstructure. Unfortunately, the resultant part has several undesirable features, which render it impractical for the bulk of laboratory applications for multi-well plates. This disposable vessel is designed to fit only their instrumentation, which possesses the industry standard tube-to-tube offset distance but none of the other industry standard geometry. Hence their consumable is not compatible with other existing thermal cycling instrumentation nor is it compatible with any other industry standard laboratory equipment used upstream or downstream of the thermal cycling step. The polypropylene sheet thermo-forming step results in a thin tube (approximately 0.0035″ (0.9 mm) thick wall) but a tube with little structural integrity; e.g. the tubes are easily crushed and deformed if not carefully handled. Additionally, the thermoforming process is not very flexible and does not allow for the formation of tubes with the depth required to accommodate standard sample volumes and to fit in standard thermal cycling equipment. Further, these processes do not allow the formation of geometry, e.g. rims around the mouths of the tube, to provide enhanced sealing of the tubes during thermal cycling.
As one can appreciate, the Analytic Jena product does not offer a practical solution to the market's need for a thermal cycling vessel which offers extremely thin and concentric tube walls, in a geometry essentially similar to conventional tubes, together with the other features and benefits of conventional injection moulded thermal cycling vessels. Until the disclosures of these claims there has not been a practical way to commercially produce a product that combines all these features and benefits.
While thermal cycling vessels may necessarily possess relatively complex geometry, they are considered one-time use, hence cost is a consideration. Injection moulding is a relatively economical manufacturing process for production of polymer products possessing relatively complex geometry and it also provides all the previously mentioned features and benefits so therefore is the preferred process for manufacturing thermal cycling vessels
As applied to the products described herein, the primary limit of the conventional injection moulding process is the minimum achievable wall thickness: In the case of polypropylene the minimum wall thickness that can be consistently filled without troublesome inclusions or weakly bonded “knit lines” (areas where two polymer fronts meet and marry together) is approximately 0.007 inch-0.009 inch (0.18 mm to 0.23 mm). As previously stated, manufacturers currently sell as “thin wall”, thermal cycling vessels with wall sections in this dimensional range but the products do not approach the reduction in wall thickness required to take advantage of the faster thermal cycling capabilities of the latest instrument technology.
In basic terms, conventional high pressure injection moulding is performed utilizing automated equipment which, at extremely high pressures and rates of speed, meter precise amounts of molten polymer into one or more cavities of a two piece mould, each cavity matching the negative of the geometry of the part to be moulded (with allowances for the shrink rate during cooling of the polymer used). The process steps may be generally described as:                1. Bringing the two pieces (halves) of the mould together such that the mating cavity portions in each mould half form one geometry which is the negative of the shape of the part to be formed.        2. Injection through one or more orifices (gates) in each cavity, of sufficient molten polymer to fill the cavity completely.        3. Allowing the part(s) to cool (solidify) sufficiently that it may be removed from the mould.        4. Opening the two halves of the mould such that the cooled part(s) may be removed or ejected from the cavity(-ies).        
Since the molten polymer enters the mould cavity at only the prescribed areas (gates), as it flows to fill the complex geometry of the cavity, cooling begins to occur. To completely fill the part it is important to maintain the flow of polymer in the cavity. Particularly with complex part geometry, as the polymer begins to cool at the point where it contacts the mould and its viscosity increases, greater pressure on the hottest material at the entrance (gates) of the cavity is required to maintain flow. In the case of very thin polypropylene wall sections, cooling happens very rapidly and a “soft skin” of polymer of approximately 0.0015 inch (0.038 mm) thick forms at the polymer/mould surface interface. This effectively reduces the flow area further in the thin wall area and effectively limits the achievable thickness of the thin wall to that mentioned above.
As regards internal stress within the part, the aforementioned cooling and fill pressure issues, combined with the fact that the chains of polymer molecules are often forced to align themselves in several directions simultaneously to fill the cavity, can lead to residual stresses in the finished part.
EP 1 618 954 A1 teaches a tube of steel construction which has a biologically inert interior coating, for instance polymer. Steel-containing tubes are not beneficial because of the costs and complexity related to the manufacturing process.
DE 4022792 and GB 806482 teach various methods of forming a vessel from a film of polymer by stretching the film. While the resulting product is of total polymer construction, using these techniques, the tube wall thickness has to be relatively constant over the whole product. This limits their use significantly, as the starting polymer film thickness defines the thickness range of the whole product. Also, no complex shapes can be manufactured.
U.S. Pat. No. 5,922,266 discloses a method of injection moulding, wherein the mould cavity is squeezed while injecting molten polymer into the cavity. The method aims at producing optically high quality articles such as contact lenses and plastic layers of optical discs. The optical properties of the article to be moulded are improved because of the reduced internal stresses of the product and reduced number of optical distortions due to the equalization of pressurization by the shrinking cavity and gradual cooling of the polymer. A similar method is disclosed in U.S. Pat. No. 4,707,321. Neither of the documents relate to manufacturing of vessels for biological assays or exceedingly thin object portions in general.
One special kind of plastic vessel and its manufacturing process by conventional injection moulding is disclosed in WO 2004/054715.